Figure 4. Extracellular acidification produces no change in fluorescence resonance energy transfer (FRET) when using pH-insensitive fluorescent proteins (FPs).

(A) Cartoons (left), electrophysiology (upper), and fluorescence traces (lower) from single cells transfected with the CFP, YFP, or both FPs attached to cASIC1 during pH jumps from eight into pH 8, pH 7, or pH 6. Fluorescence trace is the acceptor fluorescence signal divided by the donor. (B) Summary of change in acceptor/donor ratio during pH changes. (C) Same as in A but for pH-insensitive variants of GFP and RFP (see Materials and methods). (D) Summary of change in acceptor/donor ratio during pH changes. Symbols indicate single cells (N = 5–8 cells), error bars are SEM. Asterisks denote statistical significance with p-values of 0.028 for YFP pH 8 versus 6, 0.031 and 0.0025 for CFP-cA1-YFP pH 8 versus 7 and pH 8 versus 6, respectively.

Figure 4—figure supplement 1. N and C terminal GFP and RFP do not change fluorescence resonance energy transfer (FRET) efficiency upon extracellular acidification.

(A) Summary of green fluorescence emission divided by red fluorescence emission upon GFP excitation for the indicated clones. ** denotes p = 0.0014 and * indicates p = 0.0117 by randomization test. (B) Cartoon (left), example traces of current and fluorescence ratio (middle) and summary changes to apparent FRET (right) from the indicated. Symbols denote single cells (N = 3–7 cells per construct) and error bars are SEM.